CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

369 WITHDRAWN 370LB ANTIGEN RESPONSIVE CLONES OF CD4+ T CELLS CONTRIBUTE TO THE INTACT LATENT RESERVOIR Pilar Mendoza 1 , Julia Jackson 2 , Thiago Oliveira 1 , Christian Gaebler 1 , Victor Ramos 1 , Mila Jankovic 1 , Marina Caskey 1 , Michel Nussenzweig 1 , Lillian B. Cohn 2 1 The Rockefeller University, New York, NY, USA, 2 Chan Zuckerberg Biohub, San Francisco, CA, USA Background: Antiretroviral therapy suppresses but does not cure HIV-1 infection due to the existence of a long-lived reservoir of latently infected cells. The long half-life appears to partially result from expansion and contraction of infected CD4+ T cell clones over time. However, the mechanisms that govern this process in vivo are poorly understood. Methods: To test the hypothesis that expanded clones harboring latent proviruses respond to foreign antigens, we exposed CD4+ T cells from ART suppressed individuals to overlapping peptide pools from either a negative control protein, HIV-gag or CMV-pp65. Following overnight culture, activated CD4+ T cells from 8 donors were purified by cell sorting based on expression of two or more activation induced markers (AIM+: CD69, PD-L1, and 4-1BB). Total, live CD4+ T cells were sorted from the negative control. Integrated proviruses harbored by control and antigen responsive cells were enumerated and further characterized as intact or defective by combining quantitative PCR and next-gen sequencing. Results: The overall frequency of intact and defective proviruses contained within antigen responsive (AIM+) cells varied among individuals. We analyzed all HIV-1 sequences across all groups and identified clones of viral sequences in all participants. Seven of 8 donors harbored intact or defective clones of proviral sequences in antigen responsive cells. The clonal distribution of HIV-1 sequences found in AIM+ cells was significantly different from the negative control in 4 of 6 individuals for whomwe obtained sufficient data. Intact sequences from AIM+ cells were identical to replication competent viruses sequenced during outgrowth in 2 of 5 donors assayed. Conclusion: We show that both intact and defective HIV-1 proviruses can persist in clones of CD4+ T cells that respond to CMV and HIV antigens. The data suggests that infected clones of CD4+ T cells may respond to diverse pathogens in HIV-1 infected individuals. Their intermittent exposure to these and other antigens found in the virome and microbiome may account for the reported waxing and waning of individual clones of latently infected cells and their persistence over time. 371 SINGLE-CELL ANALYSIS OF IN VIVO HIV RESERVOIR UNCOVERS NOVEL MARKERS OF LATENT CELLS Jason Neidleman 1 , Xiaoyu Luo 2 , Julie Frouard 1 , Feng Hsiao 1 , Grace Xie 1 , Vincent Morcilla 3 , Katherine Sholtis 4 , Rebecca Hoh 1 , Ma Somsouk 1 , Peter W. Hunt 1 , Steven G. Deeks 1 , Nancie Archin 4 , Sarah Palmer 3 , Warner C. Greene 2 , Nadia R. Roan 1 1 University of California San Francisco, San Francisco, CA, USA, 2 Gladstone Institutes, San Francisco, CA, USA, 3 University of Sydney, Westmead, NSW, Australia, 4 University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Background: Direct phenotypic analysis of the in vivo latent HIV reservoir is complicated by the need to reactivate these cells ex vivo to identify them, which changes the phenotypes of the latent cells. We used CyTOF to quantitate the levels of 43 different proteins on reactivated cells from ART-suppressed, HIV- infected individuals, and implemented a bioinformatics approach to trace each reactivated cell to its original latent state. Methods: PBMCs (n=7), rectosigmoid biopsies (n=7), and lymph node aspirates (n=2) from treated individuals were phenotyped by CyTOF immediately after cell isolation, or stimulated with PMA/ionomycin or LRAs and then phenotyped. Reactivated cells were traced back to their original pre-stimulation state using the bioinformatics approach PP-SLIDE (Cavrois et al, Cell Reports 2017). Markers identified as preferentially expressed on latent cells were validated by sorting the cells and then conducting viral outgrowth assays and proviral sequencing. Results: Latent cells were non-randomly distributed amongst memory CD4+ T cells. Markers preferentially expressed on latent cells included those that were shared between donors (PD1, CCR5, CD2, CD49d, Ox40) and donor-specific ones (CXCR5, TIGIT, CCR6, CD28, CD7). Markers differentially expressed between latent cells in blood vs. tissues, and between latent cells reactivatable by different stimulation methods, were identified. Analysis of longitudinal samples suggested the phenotype of latent cells is stable over time. Multiparameter

sorting revealed that donor-shared surface markers identified by CyTOF markedly enriched for latent cells with replication-competent HIV: Tfh, already highly enriched for replication-competent HIV, was further enriched by 3 orders of magnitude using such markers. Viral sequencing analysis revealed the enriched cells to be largely clonally expanded. Conclusion: We have validated CyTOF phenotyping of reactivated latent cells paired with bioinformatics analysis by PP-SLIDE as an effective way to chart the in vivo blood and tissue HIV latent reservoir. Our results demonstrate that 1) latent cells are not randomly distributed amongst memory CD4+ T cells, 2) the phenotypes of latent cells are stable over time, 3) LRAs can target different latent cells than PMA/ionomycin, 4) there are shared as well as donor-specific surface markers of latent cells, and 5) sorting of cells based on surface markers identified by CyTOF markedly enriches for clonally-expanded latent cells with replication-competent HIV. 372 INTACT HIV GENOMES ARE ENRICHED IN MEMORY T CELLS WITH SHORT HALF-LIVES Vincent Morcilla 1 , Charline Bacchus-Souffan 2 , Katie Fisher 1 , Xiao Qian Wang 1 , Bethany A. Horsburgh 1 , Timothy E. Schlub 1 , Mark Fitch 3 , Rebecca Hoh 2 , Frederick M. Hecht 2 , Jeffrey Martin 2 , Steven G. Deeks 2 , Marc Hellerstein 3 , Joseph M. McCune 2 , Peter W. Hunt 2 , Sarah Palmer 1 1 The Westmead Institute for Medical Research, Westmead, NSW, Australia, 2 University of California San Francisco, San Francisco, CA, USA, 3University of California Berkeley, Berkeley, CA, USA Background: Future HIV curative therapies require a thorough understanding of the distribution of genetically intact HIV within T cell subsets during short- term antiretroviral therapy (ART) and the cellular mechanisms which maintain this reservoir. Therefore, we genetically characterized HIV genomes within T cell subsets from participants on <4 years of therapy. Methods: Seven participants were treated for <4 years either within 5 months (early, n=4) or after 7 months (late, n=3) of HIV infection. Near full-length proviral sequences were obtained from naïve (NV), stem-cell memory (SCM), central memory (CM), transitional memory (TM), effector memory (EM), and terminally differentiated (TD) CD4+T cells. Clusters of ≥2 proviral sequences which were 100% genetically identical and indicative of host cell proliferation were identified. Cellular half-lives were measured by in vivo incorporation of deuterium into genomic DNA within these cell subsets. Results: A total of 893 sequences were isolated; 585 and 308 from the early and late ART participants respectively. From these 893 sequences, 57 were considered intact (6.4%); 13 and 44 respectively from the early and late ART groups. The proportion of intact sequences across the T cell subsets was different (p=0.03). In the late ART group, the intact sequences were concentrated in cells with shorter half-lives such as TM (6/10 6 TM cells; median half-life: 95 days) and EM cells (25/10 6 EM cells; median half-life: 82 days) compared to other subsets with longer half-lives (median half-lives: 162-1107 days for NV, SCM, CM, and TD cells). For the early and late ART groups, a correlation was found where cells with shorter half-lives contained more intact proviruses (p=0.03). For the early ART participants, the clusters of identical sequences were less frequent when compared to the late ART participants (p=0.006). However, the levels of identical sequences contributing to a cluster were highest within EM and TD in all participants (p<0.001). Conclusion: The distribution of HIV genomes across T cell subsets during short-term therapy after both early and late ART suggests that a short cellular half-life could be a predictor of a higher frequency of intact proviruses. Both TD and EM cell subsets were marked by clusters of identical HIV genomes reflecting cellular proliferation. This indicates that specific cellular mechanisms such as a short half-life and greater proliferative potential, characteristics of EM T cells, contribute to the maintenance of intact HIV. 373 “FALSE ART FAILURE” FROM IDENTICAL HYPERMUTATED HIV NUCLEIC ACID IN PLASMA Johannes C. Botha 1 , Kim Steegen 2 , Lucia Hans 2 , Alan Karstaedt 3 , Sergio Carmona 3 , Denasha Reddy 4 , Mary F. Kearney 5 , John W. Mellors 6 , Gert U. van Zyl 1 1 Stellenbosch University, Tygerberg, South Africa, 2 National Health Laboratory Service, Johannesburg, South Africa, 3 University of the Witwatersrand, Johannesburg, South Africa, 4 Chris Hani Baragwanath Hospital, Johannesburg, South Africa, 5 National Cancer Institute, Frederick, MD, USA, 6 University of Pittsburgh, Pittsburgh, PA, USA

Poster Abstracts

CROI 2020 128